1. Field of the Invention
The invention relates generally to compositions and methods for binding and optically sensing anions, cations, and neutral species. Analytical methods for such species is the primary goal of optical sensing. These methods may be qualitative or quantitative. In particular, compositions containing pyrroles as the key recognition element and a quinoxaline backbone as part of the compound, are shown to provide a system with a built-in optical probe for selective sensing.
2. Description of Related Art
In the recent decades, supramolecular chemists have devoted considerable effort to developing systems capable of recognizing, sensing, and transporting anions (Dietrich, et al., 1997). This is an area of effort that is considered both timely and important. Indeed, some 70 to 75% of all natural biological processes are thought to involve a negatively charged species (Schmidtchen, 1988).
Anion recognition constitutes an important problem area within the generalized field of supramolecular chemistry. Not surprisingly, therefore, it has been pursued extensively, particularly within the calixarene domain. Indeed, most attention has focused on calixarene systems that have been modified, via attachment to, or reaction with, electron deficient metal centers, so as to make more electrophilic the normally xcfx80-electron rich calixarene moiety.
Anions constitute key components in food stuffs (e.g., fluoride, citrate and benzoate) and are products for, and pollutants from, modern agriculture (e.g., phosphate and nitrate) and can also act as potent toxins (e.g. cyanide). One anion, pertechnetate, is critical to radio-diagnostic and therapy procedures and, in a different isotopic form, is a major radioactive pollutant. Given these few examples, it is clearly important that we have a means to readily monitor the presence of these species in our everyday environment.
Among the range of biologically important anions, fluoride is of particular interest due to its established role in preventing dental caries (Kirk, 1991). Fluoride anion is also being explored extensively as a treatment for osteoporosis, (Riggs, 1984 and Kleerekoper, 1998) and, on a less salubrious level, can lead to fluorosis, (Wiseman, 1970 and Gale, et al., 1996) a type of fluoride toxicity that generally manifests itself clinically in terms of increasing bone density. This diversity of function, both beneficial and otherwise, makes the problem of fluoride anion detection one of considerable current interest. Thus, while traditional methods of fluoride anion analysis, involving, e.g., ion selective electrodes and 19F-NMR spectroscopy remain important, there is an increasing incentive to find alternative means of analysis, including those based on the use of specific chemosensors. Particularly useful would be systems that can recognize fluoride anion in solution and signal its presence via an easy-to-detect optical signature.
In the past few years, a wide range of anion sensors have been proposed (sapphyrins, Sessler, et al. 1997; calixpyrroles, Gale, et al. 1996 and Sessler, et al., 1998; cyclic polyamines, Dietrich, et al., 1981; Hoseini and Lehn, 1988) guanidinium (Dietrich, et al., 1981 and Metzger, et al., 1997)) that present varying degrees of affinity (and selectivity) toward anions such as Fxe2x88x92, Clxe2x88x92, H2PO4xe2x88x92 and/or carboxylates. Unfortunately, and in spite of considerable effort, a need for good anion sensors remains. The number of anion sensors which can select for one biological anion over a range of anions present in vivo (phosphate, chloride, fluoride, etc.) remains at best, very limited. While there exists small molecule sensors which can bind anions relatively well, they do so with little or no specificity. This is particularly true in the case of fluoride anion where few, if any, easy-to-use signaling agents exist.
In addition to anion sensing, it is also desirable to develop sensing elements capable of sensing cations and neutral species. The presence or absence, as well as the level of, various neutral molecular species is a useful diagnostic tool that can signal chemical decomposition. One example is the sensing of cis-3-hexenal (or chemical derivatives thereof), a metabolite of the bacterial E. Coli, Salmonela, and Lysteria. Such sensors would find applicability in the food industry as detectors of food contamination and spoilage. They could, for instance, be incorporated into food packaging materials.
Therefore, a need exists to develop methods and compositions for the selective detection of anions, cations, and neutral species in general, and for fluoride in particular. A motivation for the preparation of new sensors is to obtain sensor compounds designed to recognize selectively a particular analyte within a range of species and produce an easily detected signal.
The present invention provides novel compounds containing both pyrrole-derived anion and neutral species recognition subunits and an aromatic core as the optical or visual signaling group to provide chemosensors that allow for the convenient, color-based sensing of anions. Most commonly, the aromatic core will be a quinoxaline moiety, but may be any aryl system having two pyrroles covalently bound to neighboring (but not necessarily directly ajacent) carbons on an aryl moiety through a Cxe2x80x94C single bond connecting pyrroles and the aromatic moiety.
Formula I illustrates the general pyrrole-aryl systems (xcex1,xcex1 and xcex2,xcex2 substitution on the pyrrole rings) along with the specific pyrrole-quinoxaline analog shown directly below. Note that the pyrrole substitution may also be mixed, i.e, xcex1,xcex2 or xcex2,xcex1. As used herein, xe2x80x9carylxe2x80x9d means any aromatic system consisting of one or more rings which may be homonuclear or heteronuclear, and which may or may not contain aromatic or non-aromatic side groups (substitution), and which may be further complexed to one or more metals. The present invention further provides methods of use and synthetic schemes for these novel compounds. 
The present invention provides novel compounds exemplified by the pyrrolic nitrogens used as anion recognition elements with an aromatic core as a signal group. The compounds of the present invention are termed pyrrole-aryls, and as used herein, the compounds of the present invention which, at least, combine these two elements will be referred to as such. Although not shown above, the pyrrole carbon atoms may also be substituted. The aryls may or may not contain heteroatoms. Subsituents may include, but are not limited to, hydrogen, alkyl, hydroxyalkyl, glycol, polyglycol, amino, nitro, halo, cyano, aryl, heteroaryl, thio, thioalkyl, amide, ester, acyl, or carboxy and may be the same or different at each occurrence.
Compounds of the present invention may be prepared by a condensation between a 1,2-diamine and a 2,3-dipyrryl ethanedione as shown in Scheme 1. 
While specific substituents are listed above, the quinoxaline analogs may have a wider variability of substituent groups. R1 and R2 may be, individually at each occurrence, hydrogen, alkyl, hydroxyalkyl, glycol, polyglycol, amino, nitro, halo, cyano, aryl, heteroaryl, thio, thioalkyl, amide, ester, acyl, or carboxy. Although not shown above, any or all of these possible substitutions may be present on the remaining available carbon atoms of the quinoxaline. Additionally, any or all of these same possible substitution combinations may also be present on the xcex1 or xcex2 positions, or on both the xcex1 and xcex2 positions (relative to nitrogen) of the pyrrole rings.
Oxalyl chloride, o-phenylenediamine, 4-nitro-1,2-diaminobenzene were purchased from Aldrich and used without further purification. 4,5-Diamino-1,2-dimethoxybenzene was prepared according to the method of Sessler, 1992. 4,5-Dinitro-1,2-diaminobenzene was prepared according to the method of Cheeseman, 1962.
Thus, in a second respect, the present invention is the 2,3-dipyrryl-ethanediones used to produce the pyrrole-aryls. In this aspect of the invention the dipyrryl-ethanediones are of Formula II: 
wherein individually at each occurrence, each of R1-R6 are the same or different and are hydrogen, alkyl, hydroxyalkyl, glycol, polyglycol, amino, nitro, halo, cyano, aryl, heteroaryl, thio, thioalkyl, amide, ester, acyl, or carboxy. Though not shown the di-xcex2-linked diketone (bridging group attached to pyrroles in position xcex2 to nitrogen atoms) is within this family, as is the mixed xcex1, xcex2-linked diketone.
These dipyrryl-ethanediones may be produced by reaction of a pyrrol either commercially available or obtainable through synthetic methods known to one of skill in the art, with oxalyl chloride as represented in Scheme 1 to generate a variety of dipyrryl-ethanediones.
Further to this, the present invention provides a new set of novel dione compounds generated from the reaction of bipyrroles, terpyrroles etc. with oxalyl chloride to generate the compounds of Formula III: 
wherein individually at each occurrence, each of R1-R7 are the same or different and are hydrogen, alkyl, hydroxyalkyl, glycol, polyglycol, amino, nitro, halo, cyano, aryl, heteroaryl, thio, thioalkyl, amide, ester, acyl, or carboxy and n=0-10. The analogous Rx groups on either side of the diketone bond may be the same or different (i.e., R1, R2, . . . Rx on one side of the diketone bond may be the same or different from the corresponding R1, R2, . . . Rx on the opposite side, etc.; additionally, the R4 and R5 groups may have variability amongst individual pyrrole subunits; e.g. R4 on any given subunit may be the same or different from a corresponding R4 on any other subunit). Symmetry in substitution along the axis bisecting the diketone bond or among any pyrrole subunit is not required and maximum variability in substitution is possible so long as the general formula is followed.
These novel diones may then be used to generate novel pyrrole-aryl compounds such as 2,3-di(bipyrryl)quinoxalines (n=0), 2,3-di(terpyrrylquinoxalines (n=1), 2,3-di(tetrapyrrylquinoxaline (n=2) etc. The preferred route is via a condensation reaction involving the two ketones with an aryl compound.
It is further contemplated that the 2,3-dipyrryl-ethanediones may undergo reaction with any 1,2-diamine under conditions outlined in Scheme 1 to generate a variety of new compounds for anion sensing as represented by Formula IV (functionalized quinoxaline analogs) and Formula V (functionalized pyrrole, functionalized quinoxaline analogs), respectively. 
In Formula IV and V, respectively, individually at each occurrence, each of R1-R4 and R1-R10 are the same or different and are hyrdogen, alkyl, hydroxyalkyl, glycol, polyglycol, amino, nitro, halo, cyano, aryl, heteroaryl, thio, thioalkyl, amide, ester, acyl or carboxy. Note that the quinoxaline analogs are used for illustrative purposes in the above examples. It is readily apparent to one of ordinary skill in the art that an appropriate aryl or substituted aryl may be used in place of quinoxaline for the more generalized pyrrole-aryl compounds. As earlier discussed, the only requirement is orbital overlap of the ring systems comprising the aryl and pyrrole groups which are altered by bond rotation upon binding of substrate (anion, cation, or neutral atom or molecule): 
The pyrrole groups are preferably on, but need not be on, adjacent carbons of the aryl moiety. Note that the nitrogen atoms may be deprotonated to afford cation-binding systems.
2,3-dipyrrol-2xe2x80x2yl-5,6-dicyanopyrazine (Example 30) is an example of an analogous pyrrole-aryl sensing compound that does not contain the quinoxaline moiety. The present invention provides a solution to the needs described herein above by producing compounds and methods for selective sensing. In particular, the preferred pyrrole-aryl compounds of the present invention have the ability to selectively bind fluoride anion over biologically important competitors such as chloride and phosphate and in doing so, produce a color change from yellow to orange in the case of 1 and from orange to purple for 3 and 8 which is, in some circumstances, visible to the naked-eye. It was further found that for these particular analogues, organic solvents encourage fluoride binding while polar solvents, such as methanol or water, lead to fluoride dissociation. This property would allow for the original sensor to be regenerated by changing solvents once the sensing is complete.
The compounds of the present invention are particularly contemplated for use in fluoride sensing, especially in the presence of other biologically common anion species. While analogues such as 3 may display other anion sensitivities, the ability to selectively sense fluoride anion would be particularly useful for many purposes as further discussed in Examples 40 and 42.
Therefore, an aspect of the present invention is the development of analytical methods for species which are selectively bound by the pyrrole-aryl compounds. As used herein, xe2x80x9canalysisxe2x80x9d means both quantitative and qualitative analysis. As used herein, optical methods included instrumental spectroscopic methods as well as visual observation. While the focus is on optical and visual analytical methods, electrochemical methods employing the pyrrole-aryls as sensing elements are also envisioned. Time-based analytical methods, such as those monitoring changes in fluorescence lifetimes (as well as other photophysical temporal phenomena) measurements are also envisioned. Either of these analytical examples would be sensitive to the modification of the molecular electronic structure of the sensing compound which would be caused upon substrate binding. Many other analytical measurements sensitive to such changes in electronic structure and which are known to those of skill in the art would be applicable in the present invention.
It is contemplated that the pyrrole-aryls of the present invention have a wide variety of uses. A range of compounds with a large number of substituents fall within the scope of the present invention. The precursor molecules, the starting pyrrole or dione, may be derivatized as desired or the pyrrole-aryl may be modified post synthetically to yield compounds with desired substituents. Therefore, it is contemplated that the selectivity of the pyrrole-aryl compounds of the present invention will have a number of different selectivities achieved by variation of substituents within the structure.
It is also envisioned that the binding and sensing capabilities of the pyrrole-aryls can be further exploited by surface immobilization. Functionalization of the pyrrole-aryl with reactive groups would afford the ability to attach them to solid phases. Polymer phases, silica and polystyrene, among others, are solid surfaces which find applicability in this embodiment of the invention. Surface immobilization is useful in the separation sciences, in the fabrication of sensors such as fiber optic probes, as well as other applications. In the field of separation science, surface immobilization can be used to fabricate novel stationary phases. In fiber optic sensing application, immobilization of the sensing element on the distal end of a fiber optic tip can be used to construct sensors useful for remote analyses. Fiber optic sensors are known to be amenable to remote sensing, such as in vivo, in vitro or in-situ sensing. In vivo applications would involve miniaturization of the fiber optic tip, thus the high sensitivity achievable with the pyrrole-aryls is particularly advantageous. In the area of foodstuff analysis, surface immobilization of the pyrrole-aryls onto fiber optic sensors, or alternatively, onto packaging components of foodstuffs is envisioned to afford a quick, convenient way to monitor spoilage.
Additionally, the pyrrole-aryls of the present invention may be modified to increase aqueous solubility for use analytically or as therapeutic agents. In sensing applications, modification of solubility may be used to optimize a sensing element for the particular environment to be interrogated. This is performed through functionalization of the pyrrole-aryl compounds with groups that impart water solubility. Polar groups, especially those which readily carry a charge under various conditions, are candidates for such functionalization. Carboxy, hydroxy, and amine groups are most obvious but others are possible. Enhancing water solubility is useful therapeutically by enhancing bioavailability.
Additional modifications are envisioned in which the pyrrole-aryls may be incorporated into macrocyclic compound. Porphyrin-type complexes are but one example further described below. By incorporating the binding site into a macrocyclic compound, novel compounds may be made to optimize transport of therapeutic agents, or to tailor the sensing element for a specific application. Metal-linked systems of pyrrole-aryls are another aspect of the present invention. These may be prepared by first preparing silyl derivatives having, for example, TMS groups appended. Subsequent deprotection and reaction with a metal salt will afford metal linked systems.
Therapeutic uses of the compounds of the invention are also described. The binding capabilities may be exploited for uses as transporting agents. Anionic, cationic, and neutral species, through binding to the appropriate pyrrole-aryl, can be directed in vivo to areas where their therapeutic effect is optimally realized. The high affinity for a number of these species for chloride ion has potential applicability in the treatment of cystic fibrosis. Cystic fibrosis is characterized by a reduced ability to effect chloride ion transport at the cellular level involves the localized introduction of chloride ion. A means for enhancing the transport of chloride ion is therefore useful in such treatments. While this is one specific example, the ability to transport therapeutically active agents is expected to have wider applicability. This has the beneficial advantages of allowing for more efficient and lower dosages, which minimizes side and toxicity effects.
The compounds of the present invention provide a further advantage in the ease with which the pyrrole-aryls can, in light of the present disclosure, be modified. The synthetic steps are relatively simple and inexpensive to carry out. As the optical and binding properties can be controlled by the types of substituents present, this allows enough flexibility to accommodate a number of applications as well as the fine tuning of desired properties, for application in a specific environment.